Infectious viruses can be readily transmitted via products derived from biological sources or instruments used in medical procedures. By way of example, viruses may be transmitted via products and materials derived from blood products. Surgical instruments, which instruments are not adequately disinfected, have long been recognized as a means of viral transmission.
Viruses commonly associated with infection from products derived from blood products include hepatitis B virus (HBV), as well as non-A and non-B hepatitis (NANBHV), and the human immuno-deficiency virus, associated with human immuno-deficiency syndrome (AIDS). Products derived from blood pose special problems. Methods and compositions commonly used to disinfect materials tend to denature proteins, rendering the product useless. There is a need for methods and compositions that inactivate viruses in products and materials derived from biological materials and materials intended for human use.
A virion is an individual infectious particle or agent. The term xe2x80x9cvirusxe2x80x9d is used to denote a group of individual virions having common features in the nature of a species. The term xe2x80x9cvirusesxe2x80x9d is used to denote more than one species.
As used herein, the term xe2x80x9cinactivatexe2x80x9d with respect to viruses means rendering one or more individual virions unable to infect its normal host cell or replicate in its normal host cell. The term specifically excludes the removal or isolation of one or more virions from the materials in which it is found. Indeed, in accordance with the present invention, the constituents of the virion may remain in the materials in which the virion is present. As used herein, the term xe2x80x9cconstituentsxe2x80x9d refers to the proteins, peptides, fats and waxes, and nucleic acid which comprise the virion particle. Viral activity or inactivity is usually measured by testing for the development of colonies in a suitable host culture. The development of plaques of dead cells indicates the presence of the virus and its ability to infect host cells and replicate.
As used herein, the term xe2x80x9csamplexe2x80x9d refers to the materials that need to be processed to reduce the number of virions. The sample may be materials derived from biological materials, such as, by way of example, blood products, tissues, cell cultures and the like. Or, in the alternative, the sample may be a instrument or implement for which the presence of virions may pose a danger or problem. By way of example, surgical and or dental implements and instruments require disinfection prior to use.
Certain aspects of the present invention employ materials known as supercritical, critical and near critical fluids. A gas becomes a critical fluid at its critical temperature and at its critical pressure. A gas becomes a supercritical fluid at conditions which equal or exceed both its critical temperature and critical pressure. The critical temperature and critical pressure are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions which equal or exceed its critical temperature of 31.1.xc2x0 C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances, such as carbon dioxide, become dense phase fluids which have been observed to exhibit greatly enhanced solvating power. At a pressure of 204 atm (3000 psig) and a temperature of 40xc2x0 C., carbon dioxide has a density of approximately 0.8 g/cc and behaves much like a nonpolar organic solvent having a dipole moment of zero debyes.
A supercritical fluid displays a wide spectrum of solvating properties as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound""s solubility in a supercritical fluid by an order of magnitude or more. This feature allows for the fine tuning of solvation and the fractionation of mixed solutes.
The selectivity of non-polar supercritical fluid solvents can be enhanced by the addition of compounds known as modifiers (also known as entrainers and cosolvents). These modifiers are typically somewhat polar organic solvents such as acetone, ethanol. methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows a wide latitude in the variation of solvent power.
Supercritical fluids exhibit liquid-like density yet retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Supercritical fluids exhibit low surface tension allowing facile penetration into micro-porous materials.
A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. A material at conditions that border its supercritical state are known as xe2x80x9cnear critical fluids.xe2x80x9d For the purposes of this application, a near critical fluid is a fluid that is:
(a) at a temperature between its critical temperature (Tc) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure (Pc); or,
(b) at a pressure between its critical pressure (Pc) and 75% of its critical pressure and a temperature at least 75% of its critical temperature (measured in absolute scales of degrees Kelvin and psia).
Table 1 below sets forth physical properties of materials commonly employed as supercritical, critical or near critical fluids. To simplify the terminology, individuals skilled in the art will refer to materials which are at conditions which are supercritical or near critical as critical fluids, as xe2x80x9ccritical fluids.xe2x80x9d This application will refer to supercritical, near critical or critical fluids collectively as xe2x80x9cSCNCorCxe2x80x9d fluids.
As used above, temperature is expressed in degrees Centigrade, and pressure is expressed in psig at 25xc2x0 C. As used above BP represents normal boiling point, and PVAP represents vapor pressure.
There exists a need to render viruses, especially enveloped and lipid coated viruses in proteinaceous products, inactive without incurring substantial denaturation. In particular, there exists a need to render viruses inactive in blood derived products without changing the nature of the blood products necessary for their function.
The present invention relates to methods and apparatus for inactivating viruses associated with a sample. One embodiment of the present method relates to blood derived samples, such as plasma, serum, blood fractions, platelets and the like. The method comprises the steps of forming an admixture of a blood derived sample with a critical, near critical or supercritical fluid which critical, near critical or supercritical fluid is capable of being received by one or more virions associated with the sample. Upon removal of the critical, near critical, or supercritical fluid one or more virions are inactivated. The method further comprises the step of removing the critical, near critical or supercritical fluid to render one or more virions inactive while retaining the constituents of the virus to form a processed blood derived product. The processed blood derived product exhibits a reduction of viral activity compared with the original blood derived sample The steps of the process can be repeated to effect a desired level of inactivation of virions.
The present method has particular application for the inactivation of virus associated with blood borne illnesses, such as AIDS, in blood derived samples. Surprisingly and unexpectedly, under conditions that leave the blood derived sample substantially unchanged, a four log reduction in viral activity can be achieved. As used herein, when referring to blood derived products, the term xe2x80x9csubstantially unchangedxe2x80x9d means exhibiting negligible denaturation of blood proteins to no more than fifty percent reduction in the activity of blood proteins. The time in which the blood derived sample may be processed to achieve this four log reduction in viral activity may be as little as five minutes.
Preferably, the critical, supercritical or near critical fluid is at a temperature in the range of 0xc2x0 C. to 100xc2x0 C. This temperature range is in a range in which proteins held in aqueous solutions do not denature. Preferably, the critical, supercritical or near critical fluid has a temperature that does not exceed 60xc2x0 C. And even more preferred, the critical, super critical or near critical fluid has a range of 4xc2x0 C. to 40xc2x0 C.
Preferably, the critical, supercritical or near critical fluid has a pressure in which the admixture is made and maintained which pressure is 0.75 to 20.0 times the critical pressure of the gas comprising such fluid.
A preferred fluid is selected from one or more of the gases of the group consisting of fluorocarbons, such as chlorofluoromethanes, alkanes, such as ethylene, propane and ethane, and binary gases such as nitrous oxide and carbon dioxide. Preferably, the critical, supercritical or near critical fluid further comprises one or more modifiers selected from the group consisting of ethanol, methanol, acetone, and ethylene glycol.
A particularly preferred critical, supercritical or near critical fluid is nitrous oxide at approximately 12xc2x0 C. to 30xc2x0 C. at 800 to 1600 psig; and, even more preferred, nitrous oxide at approximately 16xc2x0 C. to 26xc2x0 C. at 1000 to 1400 psig; and, most preferred, nitrous oxide at approximately 21xc2x0 C. at approximately 1200 psig. At these conditions, blood proteins show little change in function.
A particularly preferred critical, supercritical or near critical fluid is chlorodifluoromethane at approximately 10xc2x0 C. to 60xc2x0 C. at 1000 to 4000 psig; and, even more preferred, chlorodifluoromethane at approximately 40xc2x0 C. at 2000 to 4000 psig.
One embodiment of the present invention features an apparatus for inactivating one or more virions in a blood sample. The apparatus comprises a vessel for forming an admixture of a blood derived sample with a critical, near critical or supercritical fluid which critical, near critical or supercritical fluid is capable of being received by one or more virions associated with the sample. Upon removal of the critical, near critical, or supercritical fluid one or more virions are inactivated. The apparatus further comprises depressurization means for removing the critical, near critical or supercritical fluid to render one or more virions inactive while retaining the constituents of the virus in the sample.
Preferably, the vessel is in communication with a continuous supply of the blood sample. And, the depressurization means is capable of receiving a continuous supply of the admixture of the blood sample and the critical, supercritical or near critical fluid.
Preferably, the vessel retains the admixture for a period of time to effect a thousand-fold to four thousand-fold reduction of active virions. And, more preferably, the vessel retains the admixture for a period of five to thirty minutes.
These and other benefits of the present invention will be apparent to individuals skilled in the art upon reading the detailed description and viewing the drawings.